The regulation of platelet production and function are critical determinants of cardiovascular health. Although it has been nearly a decade since the primary regulator of platelet production, thrombopoietin (TPO), was cloned and initially characterized, there remain many unanswered questions regarding how the hormone interacts with other cellular signals to affect hematopoietic stem cells (HSC), megakaryocytes (MKs) and platelets. In addition to cytokines and growth factors, a major influence on hematopoietie cells is derived from their interactions with the marrow microenvironment, mediated mostly through cell surface integrins and other adhesion molecules. We and others have shown that integrins affect TPO-induced HSC and MK growth, and TPO, in turn, affects platelet activation, mediated mostly through integrin engagement of the extracellular environment. Unfortunately, although our understanding of growth factor and integrin signaling is improving, little is known of the molecular mechanisms by which TPO and integrin signals interact. To address this question a research plan of three specific aims is proposed, designed to explore this 1) in HSCs, specifically, whether the combined effects of TPO and integrins are mediated through the induction of HoxB4 or translocation of HoxA9, two transcription factors vital for stem cell physiology, or through TPO induction of HSC vascular endothelial cell growth factor (VEGF expression or its receptor, molecules shown to play an important role in the self-renewal, survival or expansion of stem cells 2) in MKs, specifically, whether activation of integrin regulated kinases, including focal adhesion kinase (FAK) and Pyyk2 augment TPO induction of the secondary signaling kinases phosphoinositol 3 kinase (PI3K), protein kinase (PK)Czeta mitogen activated protein kinase (MAPK), and whether engagement of this pathway plays a role in the thrombocytosis oi inflammation, and 3) in platelets, specifically, whether TPO affects shear stress induced Ca ++ signaling, PI3K, MAPK or signaling from focal kinases to account for its effect on platelet aggregation. In each of these aims the general strategy will be to identify the "circuitry" that mediates the combined TPO/adhesion molecule effect(s), and then determine the physiological relevance of each pathway by interrogating the system in tissue culture using chemical inhibitors, ribozymes or small interfering (si)RNA, and in vivo utilizing genetic knock-out and knock-in approaches. It is anticipated that the knowledge gained by successful completion of the proposed work will directly yield new insights into how integrins can interact with ]cytokines to affect critical cellular events, and provide novel approaches to the diagnosis and treatment of clinical disorders of hemostasis and thrombosis.